The present invention relates to the use of primers in polymerase chain reaction assays for the detection of a heretofore unknown species of the banana pathogen Mycosphaerella. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations.
Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, in many parts of the world, to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite $1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981; Seed Sci. and Technol. 9: 679-685).
The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981; Proc. 1981 Brit. Crop Prot. Conf.) contended that 24% of the powdery mildew populations from spring barley and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties, with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and Mycosphaerella fijiensis to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).
There are two well-known forms of sigatoka leaf spots which affect bananasxe2x80x94yellow sigatoka, caused by Mycosphaerella musicola and black sigatoka caused by Mycosphaerella fijiensis. Black sigatoka is the more economically devastating, causing causes significant reductions in leaf area, yield losses of 50% or more, and premature ripening, a serious defect in exported fruit . It is more damaging and difficult to control than the related yellow sigatoka disease, and has a wider host range that includes the plantains and dessert and ABB cooking bananas that are usually not affected by yellow sigatoka.
In export plantations, black sigatoka is controlled with frequent applications of fungicides. This is a very expensive practice because it includes the use of airplanes or helicopters, permanent landing strips and facilities for mixing and loading the fungicides, and the high recurring expense of the spray materials themselves. In total, it has been estimated that these costs are ultimately responsible for 25% of the final retail price of these fruit in the importing countries (www.scisoc.org/feature/banana/top.html as found on Apr. 17, 2000). Different sterol demethylation inhibitors (DMIs) are now the most commonly used compounds for the control of sigatoka, but increased tolerance of the pathogen to the DMI fungicides has made it necessary to increase applications in several countries in banana-growing regions to frequencies of 25-40 per year (www.scisoc.org/feature/banana/top.html as found on Apr. 17, 2000).
Although black sigatoka can often be recognized visually, unambiguous diagnosis can be complicated by the presence of other pathogens found on banana leaves. Isolation of the pathogen, which is most successfully achieved by ascospore discharge from necrotic leaf material, is often confounded by the absence of mature perithecia, and even when obtained in culture, I M. fijiensis and M. musicola are not readily visually differentiated (Johanson and Jeger Mycol. Res. 97 (6): 670-674 (1993)).
Biomedical researchers have used PCR-based techniques for some time and with moderate success to detect pathogens in infected animal tissues. More recently, however, this technique has been applied to detect plant pathogens. The presence of Gaumannomyces graminis in infected wheat has been detected using PCR of sequences specific to the pathogen mitochondrial genome (Schlesser et al., 1991; Applied and Environ. Microbiol. 57: 553-556), and random amplified polymorphic DNA (i.e. RAPD) markers were able to distinguish numerous races of Gremmeniella abietina, the causal agent of scleroderris canker in conifers. U.S. Pat. No. 5,585,238 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Septoria tritici, Septoria nodorum, Pseudocercosporella herpotrichoides (R- and W-types), Mycosphaerella fijiensis, and Mycosphaerella musicola and their use in the identification of these fungal isolates using PCR-based techniques. In addition, U.S. Pat. No. 5,955,274 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Fusarium and their use in the identification of these fungal isolates using PCR-based techniques. Furthermore, U.S. Pat. No. 5,800,997 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia and their use in the identification of these fungal isolates using PCR-based techniques.
In view of the above, there is a real need for the development of technology that will allow the identification of additional specific races of pathogen fungi early in the infection process. By identifying the specific race of a pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.
The present invention pertains to methods of identification of different pathotypes of plant pathogenic fungi. The invention provides Internal Transcribed Spacer (ITS) DNA sequences that show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they are used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and is thus used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.
In particular, the present invention provides the Internal Transcribed Spacer (ITS) DNA sequences from a heretofore unknown species of Mycosphaerella, as well as ITS-derived diagnostic primers for the detection of this species of Mycosphaerella and for differentiating it from other Mycosphaerella species such as Mycosphaerella fijiensis and Mycosphaerella musicola. 
In one embodiment, the present invention provides a DNA molecule isolated from the ribosomal RNA gene region of a fungal pathogen, wherein said DNA molecule is the Internal Transcribed Spacer (ITS) DNA sequence of a heretofore unknown species of Mycosphaerella. In a preferred embodiment, the Internal Transcribed Spacer sequence from the heretofore unknown species of Mycosphaerella is selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 19.
According to another embodiment, the present invention provides an oligonucleotide primer for use in PCR-based detection of a Mycosphaerella species, wherein said primer has sequence identity with at least 10 contiguous nucleotides of the Internal Transcribed Spacer sequence from the heretofore unknown species of Mycosphaerella. Preferably, said oligonucleotide primer has a nucleotide sequence consisting essentially of the sequence set forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, or 13. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant up to 10 additional residues on either the 5xe2x80x2 or 3xe2x80x2 end, or both the 5xe2x80x2 and 3xe2x80x2 end, of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, or 13. Most preferably, the oligonucleotide primer is selected from the group consisting of SEQ ID NOs:5-13.
According to yet another embodiment, the present invention provides a pair of oligonucleotide primers for use in PCR-based detection of a Mycosphaerella species, wherein at least one of said primers is the oligonucleotide primer described in the preceding paragraph. Preferably, the pair of oligonucleotide primers is selected from the group consisting of: SEQ ID NO:5 and SEQ ID NO:10; SEQ ID NO:5 and SEQ ID NO:11; SEQ ID NO:5 and SEQ ID NO:12; SEQ ID NO:5 and SEQ ID NO:13; SEQ ID NO:5 and SEQ ID NO:4; SEQ ID NO:6 and SEQ ID NO:10; SEQ ID NO:6 and SEQ ID NO:11; SEQ ID NO:6 and SEQ ID NO:12; SEQ ID NO:6 and SEQ ID NO:13; SEQ ID NO:6 and SEQ ID NO:4; SEQ ID NO:7 and SEQ ID NO:10; SEQ ID NO:7 and SEQ ID NO:11; SEQ ID NO:7 and SEQ ID NO:12; SEQ ID NO:7 and SEQ ID NO:13; SEQ ID NO:7 and SEQ ID NO:4; SEQ ID NO:8 and SEQ ID NO:10; SEQ ID NO:8 and SEQ ID NO:11; SEQ ID NO:8 and SEQ ID NO:12; SEQ ID NO:8 and SEQ ID NO:13; SEQ ID NO:8 and SEQ ID NO:4; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:9 and SEQ ID NO:11; SEQ ID NO:9 and SEQ ID NO:12; SEQ ID NO:9 and SEQ ID NO:13; SEQ ID NO:9 and SEQ ID NO:4; SEQ ID NO:1 and SEQ ID NO:10; SEQ ID NO:1 and SEQ ID NO:11; SEQ ID NO:1 and SEQ ID NO:12; and SEQ ID NO:1 and SEQ ID NO:13. Most preferably, the pair of oligonucleotide primers is SEQ ID NO: 7 and SEQ ID NO: 12.
According to still another embodiment, the present invention provides a method for the detection of a Mycosphaerella species, comprising: (a) isolating DNA from plant tissue infected with said Mycosphaerella species; (b) amplifying a part of the Internal Transcribed Spacer sequence of said Mycosphaerella species using said DNA as a template in a polymerase chain reaction with a pair of the above-described primers; and (c) detecting said Mycosphaerella species by visualizing the amplified part of the Internal Transcribed Spacer sequence.
This invention provides the possibility of assessing potential damage in a specific crop variety-pathogen strain relationship and of utilizing judiciously the diverse armory of fungicides that is available. Furthermore, the invention can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas. The invention provides a method of detection that is especially suitable for diseases with a long latent phase.
Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of a Mycosphaerella fungal pathogen.
SEQ-ID-NO:1 Oligonucleotide Primer ITS1.
SEQ-ID-NO:2 Oligonucleotide Primer ITS2.
SEQ-ID-NO:3 Oligonucleotide Primer ITS3.
SEQ-ID-NO:4 Oligonucleotide Primer ITS4.
SEQ-ID-NO:5 Oligonucleotide Primer J-BP1.
SEQ-ID-NO:6 Oligonucleotide Primer J-BP2.
SEQ-ID-NO:7 Oligonucleotide Primer J-BP3.
SEQ-ID-NO:8 Oligonucleotide Primer J-BP4.
SEQ-ID-NO:9 Oligonucleotide Primer J-BP5.
SEQ-ID-NO:10 Oligonucleotide Primer J-BP6.
SEQ-ID-NO:11 Oligonucleotide Primer J-BP7.
SEQ-ID-NO:12 Oligonucleotide Primer J-BP8.
SEQ-ID-NO:13 Oligonucleotide Primer JB-473.
SEQ-ID-NO:14 Truncated DNA sequence for the Internal Transcribed Spacer of a fungus amplified from banana sample xe2x80x9cCapesterre-babin 2xe2x80x9d (pCRBPCapbabB2-1).
SEQ-ID-NO:15 Truncated DNA sequence for the Internal Transcribed Spacer of a fungus amplified from banana sample xe2x80x9cMatouba bas 3xe2x80x9d (pCRBPMatbasB3-2).
SEQ-ID-NO:16 Truncated DNA sequence for the Internal Transcribed Spacer of a fungus amplified from banana sample xe2x80x9cTemoin Infest Fortexe2x80x9d (pCRBPMf9).
SEQ-ID-NO:17 DNA sequence for the Internal Transcribed Spacer of Mycosphaerella fijiensis ATCC isolate #22116 (U.S. Pat. No. 5,585,238).
SEQ-ID-NO:18 DNA sequence for the Internal Transcribed Spacer of Mycosphaerella musicola ATCC isolate #22115 (U.S. Pat. No. 5,585,238).
SEQ-ID-NO:19 Consensus sequence of Internal Transcribed Spacer DNA of fungi amplified from three infected banana samples (Consensus of SEQ ID NOs: 14-16).
The present invention provides unique DNA sequences that are useful in identifying different pathotypes of plant pathogenic fungi. Particularly, the DNA sequences can be used as primers in PCR-based analysis for the identification of fungal pathotypes. The DNA sequences of the invention include the Internal Transcribed Spacer (ITS) sequences of the ribosomal RNA gene region of a particular Mycosphaerella fungal pathogen as well as primers derived from this region that are capable of identifying this Mycosphaerella pathogen. ITS DNA sequences from different pathotypes within a pathogen species or genus, which vary between the different members of the species or genus, can be used to identify those specific members.
Ribosomal genes are suitable for use as molecular probe targets because of their high copy number. Despite the high conservation between mature rRNA sequences, the non-transcribed and transcribed spacer sequences are usually poorly conserved and are thus suitable as target sequences for the detection of recent evolutionary divergence. Fungal rRNA genes are organized in units, each of which encodes three mature subunits of 18S (small subunit), 5.8S, and 28S (large subunit). These subunits are separated by two Internal Transcribed Spacers, ITS1 and ITS2, of around 300 bp (White et al., 1990; In: PCR Protocols; Eds.: Innes et al.; pages 315-322). In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs). ITS and NTS sequences are particularly suitable for the detection of specific pathotypes of different fungal pathogens.
The DNA sequences of the invention are from the Internal Transcribed Spacer sequences of the ribosomal RNA gene region of a heretofore unknown species of a Mycosphaerella plant pathogen. The ITS DNA sequences from different pathotypes within a pathogen species or genus vary among the different members of the species or genus. Once having determined the ITS sequences of a pathogen, these sequences can be aligned with other ITS sequences, such as those from M. fijiensis and M. musicola. In this manner, primers can be derived from the ITS sequences. That is, primers can be designed based on regions within the ITS sequences that contain the greatest differences in sequence among the fungal pathotypes. These sequences and primers based on these sequences can be used to identify specific pathogens.
Sequences of representative oligonucleotide primers derived from the Mycosphaerella ITS sequences of the invention are disclosed in SEQ ID NOs:5-13. The sequences find use in the PCR-based identification of the pathogens of interest.
Methods for the use of the primer sequences of the invention in PCR analysis are well known in the art. For example, see U.S. Pat. Nos. 4,683,195 and 4,683,202, as well as Schlesser et al. (1991) Applied and Environ. Microbiol. 57:553-556. See also, Nazar et al. (1991; Physiol. and Molec. Plant Pathol. 39: 1-11), which used PCR amplification to exploit differences in the ITS regions of Verticillium albo-atrum and Verticillium dahliae and therefore distinguish between the two species; and U.S. Pat. No. 5,585,238, which describes similar techniques to distinguish the banana pathogens Mycosphaerella fijiensis and Mycospharella musicola. 
The ITS sequences are compared within each pathogen group to locate divergences that might be useful to test in PCR to distinguish the different species and/or strains. From the identification of divergences, numerous primers are synthesized and tested in PCR-amplification. Templates used for PCR-amplification testing are firstly purified pathogen DNA, and subsequently DNA isolated from infected host plant tissue. Thus, it is possible to identify pairs of primers that are diagnostic, i.e. that identify one particular pathogen species or strain but not another species or strain of the same pathogen. Primers are also designed to regions highly conserved among the species to develop genus-specific primers as well as primers that will identify any of several fungal pathogens that cause a particular disease. For example, as described herein, primers are developed to differentiate a heretofore unknown species of Mycosphaerella from other species of Mycosphaerella such as M. fijiensis and M. musicola. 
Preferred primer combinations are able to distinguish between the different species or strains in infected host tissue, i.e. host tissue that has previously been infected with a specific pathogen species or strain. This invention provides numerous primer combinations that distinguish the heretofore unknown species of Mycosphaerella from other species of Mycosphaerella such as M. fijiensis and M. musicola. The primers of the invention are designed based on sequence differences among the fungal ITS regions. A minimum of one base pair difference between sequences can permit design of a discriminatory primer. Primers designed to a specific fungal DNA""s ITS region can be used in combination with a primer made to a conserved sequence region within the ribosomal DNA""s coding region to amplify species-specific PCR fragments. In general, primers should have a theoretical melting temperature between about 60xc2x0 C. to about 70xc2x0 C. to achieve good sensitivity and should be void of significant secondary structure and 3xe2x80x2 overlaps between primer combinations. Primers generally have sequence identity with at least about 5-10 contiguous nucleotide bases of ITS1 or ITS2. In preferred embodiments, primers are anywhere from approximately 5 to 30 nucleotide bases long.
The present invention lends itself readily to the preparation of xe2x80x9ckitsxe2x80x9d containing the elements necessary to carry out the process. Such a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more containers, such as tubes or vials. One of the containers may contain unlabeled or detectably labeled DNA primers. The labeled DNA primers may be present in lyophilized form or in an appropriate buffer as necessary. One or more containers may contain one or more enzymes or reagents to be utilized in PCR reactions. These enzymes may be present by themselves or in admixtures, in lyophilized form or in appropriate buffers.
Finally, the kit may contain all of the additional elements necessary to carry out the technique of the invention, such as buffers, extraction reagents, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like.
The examples below show typical experimental protocols that can be used in the selection of suitable primer sequences, the testing of primers for selective and diagnostic efficacy, and the use of such primers for disease and fungal isolate detection. Such examples are provided by way of illustration and not by way of limitation.